This invention relates to the field of prostate cancer detection. More specifically, novel compositions are provided which serve as prognostic indicators for late stage disease. Methods are also provided which facilitate the identification of those patients at risk for aggressive prostate cancer progression.
Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
This year prostate cancer is expected to be diagnosed in 200,000 men in the U.S. and to result in the loss of 38,000 lives. Such numbers make prostate cancer the most frequently diagnosed malignancy (other than that of the skin) in American males and the second leading cause of cancer-related death in that group. Physicians usually detect cancers by finding a lump in the prostate gland, which is a walnut shaped structure that helps to maintain the viability of sperm. Such lumps may be discovered during a routine checkup or an examination prompted by a patient""s complaint of sudden urinary discomfort, or occasional impotence.
In some instances, prostate cancer is detected in the course of treatment for a disorder called benign prostatic hyperplasia. This condition, an aging-related enlargement of the prostate, affects more than half of all men older than 45 and gives rise (albeit more gradually) to the same urinary troubles caused by a prostate tumor. If the symptoms become too troublesome, a transurethral resection of the prostate, a process whereby parts of the gland are scraped away may be performed. Whenever resection is done, the excised tissue is analyzed under a microscope for evidence of malignancy, which is occasionally found.
A simple blood test for prostate specific antigen (PSA) constitutes a third means of detecting prostate cancer. Increased PSA levels can signal the presence of cancer in individuals who display no symptoms of prostate abnormalities.
Most prostate cancer (CaP) patients have no known risk factors for tumor development or rate of disease progression. The present inventors have appreciated the need for molecular markers for prostate cancer progression to identify patients who are at risk for aggressive disease and would benefit from early treatment.
This invention provides novel biological molecules useful for identification, detection and/or regulation of complex signaling events involved in prostate cancer progression. According to one aspect of the present invention, an isolated double stranded nucleic acid molecule, CLAR1, is provided which encodes a protein between about 250 and about 300 amino acids in length (preferably about 276 amino acids) that is a late stage specific marker for prostate cancer progression. The protein encoded by the CLAR1 nucleic acid molecule comprises a presently determined carboxy-terminal serine phosphorylation site and at least one or a multiplicity of SH3 binding domains. In a particularly preferred embodiment, the CLAR1 marker protein has an amino acid sequence of SEQ ID NO: 2. An exemplary nucleic acid molecule of the invention is set forth in SEQ ID NO: 1.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) SEQ ID NO: 1; (2) a sequence which hybridizes with SEQ ID NO: 1; 3) a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 2; 4) a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 3; and 5) a natural allelic variant of a sequence of 1), 2), 3) or 4).
According to another aspect of the present invention, an isolated late stage-specific prostate cancer progression marker protein is provided which has a deduced molecular weight of between about 30 kDa and 50 kDa (preferably between about 30 kDa and 40 kDa and most preferably 33.8 kDa). In a preferred embodiment of the invention, the protein is of human origin, and has an amino acid sequence which is the same as or substantially the same as SEQ ID NO: 2. In yet another embodiment, the polypeptide may be derived from an alternatively spliced CLAR1 mRNA molecule and has a sequence the same as or substantially the same as SEQ ID NO: 3.
A further aspect of the present invention provides an oligonucleotide or polynucleotide fragment of the nucleotide sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, in particular, for use in a method of obtaining and/or screening nucleic acid.
According to another aspect of the present invention, antibody binding domains or antibodies immunologically specific for the proteins described hereinabove are provided.
Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specifications and claims. The terms xe2x80x9cspecifically hybridizing,xe2x80x9d xe2x80x9cpercent similarityxe2x80x9d and xe2x80x9cpercent identity (identical)xe2x80x9d are defined in detail in the description set forth below.
With reference to nucleic acids of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5xe2x80x2 and 3xe2x80x2 directions) in the naturally occurring genome of the organism in which it originated. For example, the xe2x80x9cisolated nucleic acidxe2x80x9d may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. A nucleic acid molecule of the present invention may be single or double stranded.
With respect to RNA molecules of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a xe2x80x9csubstantially purexe2x80x9d form (the term xe2x80x9csubstantially purexe2x80x9d is defined below).
With respect to protein, the term xe2x80x9cisolated proteinxe2x80x9d or xe2x80x9cisolated and purified proteinxe2x80x9d is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in xe2x80x9csubstantially purexe2x80x9d form.
The term xe2x80x9csubstantially purexe2x80x9d refers to a preparation comprising at least 50-60% by weight of a given compound (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like). xe2x80x9cIsolated is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into pharmaceutically acceptable preparations.
With respect to antibodies of the invention, the term xe2x80x9cimmunologically specificxe2x80x9d refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., CLAR1), but which do not substantially recognize and specifically bind other molecules in a sample containing a mixed population of antigenic biological molecules.
With respect to oligonucleotides, the term xe2x80x9cspecifically hybridizingxe2x80x9d refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed xe2x80x9csubstantially complementaryxe2x80x9d). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
An oligonucleotide is preferably at least 10 nucleotides in length, more preferably at least 15 nucleotides in length, most preferably at least 20 nucleotides in length.
The present invention also includes active portions, fragments, derivatives and functional mimetics of the CLAR1 polypeptide or protein of the invention.
An xe2x80x9cactive portionxe2x80x9d of CLAR1 polypeptide means a peptide which is less than said full length CLAR1 polypeptide, but which retains its essential biological activity.
A xe2x80x9cfragmentxe2x80x9d of the CLAR1 polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. Fragments of the CLAR1 polypeptide sequence, antigenic determinants or epitopes are useful for raising antibodies to a portion of the CLAR1 amino acid sequence.
A xe2x80x9cderivativexe2x80x9d of the CLAR1 polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the essential activity of the wildtype CLAR1 polypeptide.
xe2x80x9cFunctional mimeticxe2x80x9d means a substance which may not contain an active portion of the CLAR1 amino acid sequence, and probably is not a peptide at all, but which retains the essential biological activity of natural CLAR1 polypeptide.
As outlined above, the CLAR1 polypeptide or protein of the invention includes any analogue, fragment, derivative or mutant which is derived from a CLAR1 polypeptide and which retains at least one property of the CLAR1 polypeptide. Different xe2x80x9cvariantsxe2x80x9d of the CLAR1 polypeptide exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve different splicing or post-translational modification. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include inter alia: (a) variants in which one or more amino acids residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the CLAR1 polypeptide, (c) variants in which one or more amino acids includes a substituent group, and (d) variants in which the CLAR1 polypeptide is fused with another polypeptide such as serum albumin. Other CLAR1 polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations etc), chemical, and enzymatic techniques that are known to the person having ordinary skill in the art.
If such allelic variations, analogues, fragments, derivatives, mutants, and modifications, including alternative mRNA splicing forms and alternative post-translational modification forms result in derivatives of the CLAR1 polypeptide which retain any of the biological properties of the CLAR1 polypeptide, they are included within the scope of this invention.
In a further aspect of the present invention, there is provided a kit for detecting CLAR1 nucleic acid according to the present invention associated with cancer, or a susceptibility to cancer, the kit comprising one or more nucleic acid probes capable of binding and/or detecting a CLAR1 nucleic acid. Alternatively, the kit may comprise one or more antibodies capable of specifically binding and/or detecting CLAR1 nucleic acid or protein or a pair of oligonucleotide primers having sequences corresponding to, or complementary to a portion of the nucleic acid sequence set out in SEQ ID NO: 1 for use in amplifying a CLAR1 nucleic acid by, for example, polymerase chain reaction (PCR).
In yet another aspect of the invention, transgenic animals, including CLAR1 knock-out animals, are provided which are useful for elucidating the role of CLAR1 plays in neonatal development and cancer progression.
There is currently a need for models of prostate cancer, including animal models, to enable screening and identification of compounds for the treatment of this disease. CLAR1 gene expression is changed during prostate cancer development and disease progression. A transgenic animal expressing the CLAR1 protein would provide a useful model in which to investigate cancer development, tumor progression, and therapeutic effects. Preferably, the transgenic animal expresses the CLAR1 protein at a level which is higher than the normal level of CLAR1 protein.
Generally speaking, the murines, namely mice, rats and guinea pigs, are the most widely used animal models for disease. They are easy to manipulate and inexpensive. Unfortunately, these small mammals are not always compatible with the intended application. Thus, they are not always representative of the human model and its metabolism. Closer to man, the chimpanzee is a test animal which is used in particular for detecting therapeutic agents and vaccines which are directed against AIDS and cancer. However, its very substantial cost constitutes a major and compelling handicap with regard to its use.
The term xe2x80x9canimalxe2x80x9d is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A xe2x80x9ctransgenic animalxe2x80x9d is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA. The term xe2x80x9cgerm cell-line transgenic animalxe2x80x9d refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals.
In a specific embodiment, a transgenic animal expresses CLAR1. This transgenic animal may be a mouse, rat, guinea pig, dog, cat, rabbit, simian, and the like.
The information may be foreign to the species of animal to which the recipient belongs, (i.e. exogenous) foreign only to the particular individual recipient (i.e. exogenous), or genetic information already possessed by the recipient. In the last case, the introduced gene may be differently expressed compared to the native endogenous gene.
The genes may be obtained by isolating them from genomic sources, by preparation of cDNAs from isolated RNA templates, by directed synthesis, or by some combination thereof.
A transgenic animal according to the invention can integrate the CLAR1 encoding DNA sequences into all its cells or only into a certain percentage of cells; in the latter case it would be termed mosaic. In general, the CLAR1 encoding DNA sequences are integrated into all the cells. The inserted DNA sequences according to the invention encode all, or an active part, of the CLAR1 protein or variant thereof.
To be expressed within the transgenic animal, a gene should be operably linked to a regulatory region. Regulatory regions, such as promoters, may be used to increase, decrease, regulate or designate to certain tissues or to certain stages of development the expression of a gene. The promoter need not be a naturally occurring promoter. The xe2x80x9ctransgenic non-human animalxe2x80x9d of the invention are produced by introducing a xe2x80x9ctransgenexe2x80x9d into the germline of the non-human animal. The methods enabling the introduction of DNA into cells are generally available and well-known in the art. Different methods of introducing transgenes could be used. Generally, the zygote is the best target for microinjection. The use of zygotes as a target for gene transfer has a major advantage. In most cases, the injected DNA will be incorporated into the host gene before the first cleavage (Brinster, et al., (1985) Proc. Nat. Acad. Sci. USA 82, 4438-4442). Consequently, nearly all cells of the transgenic non-human animal will carry the incorporated transgene. Generally, this will also result in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is a preferred method for incorporating transgenes in practicing the invention.
Retroviral infection can also be used to introduce a transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, blastomeres may be targets for retroviral infection (Jaenich, R. (1976) Proc. Nat. Acad. Sci. USA 73, 1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zone pellucida (Hogan, et al., (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., (1985) Proc. Natl. Acad. Sci. USA 82, 6927-6931; Van der Putten et al., (1985) Proc. Nat. Acad. Sci. USA 82, 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten et al., (1985) Proc. Natl. Acad. Sci. USA 82, 6148-6152; Stewart et al, (1987) EMBOJ. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al., (1982) Nature 298:623-628). Most of the founder animals will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Furthermore, the founder animal may contain retroviral insertions of the transgene at a variety of positions in the genome; these generally segregate into the offspring. In addition, it is also possible to introduce a transgene into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et al., (1982) Nature 298:623-628.).
A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro (Evans, M. J., et al., (1981) Nature 292, 154-156; Bradley, A., et al. (1984) Nature 309, 255-258; Gossler, et al., (1986) Proc. Natl. Acad Sci. USA 83, 9065-9060; and Robertson, et al., (1986) Nature 322, 445-448). Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus-mediated transduction. The resulting transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal (For review see Jaenisch, R. (1988) Science 240, 1468-1474).
The methods for evaluating the presence of the introduced DNA as well as its expression are readily available and well-known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the exogenous DNA polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.
As used herein, a xe2x80x9ctransgenexe2x80x9d is a DNA sequence introduced into the germline of a non-human animal by way of human intervention and genetic engineering.
The nucleic acids, proteins/polypeptides, peptides and antibodies of the present invention are useful as diagnostic and/or prognostic indicators for assessing patients at risk for aggressive prostate cancer. They may also be used as research tools and should facilitate the elucidation of the mechanistic action of the novel genetic and protein interactions involved in the progression of prostate cancer.
The present invention also provides nucleic acid molecules, proteins, polypeptides or antibodies, as defined above, for use in medical treatment and preferably for use in the preparation of a medicament for the treatment of cancer, in particular prostate cancer.
Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.